Brucella infects a significant number of people and livestock in developing countries and infects wild as well as domestic animals in the United States. In addition, Brucella is a potential biowarfare agent; strains of Brucella have been constructed with resistance to multiple antibiotics used to treat the disease. These strains pose a significant morbidity and mortality threat to exposed personnel. Brucellosis symptoms include recurring fever, chills and anxiety. Even though the disease is rarely fatal, once well established, the disease is difficult to treat since the bacteria reside in the bone marrow.
Two live attenuated Brucella strains currently approved for use as animal vaccines, B. abortus Strain 19 [Cheville, et al. (1993) Am. J. Vet. Res. 54:1591-1597; Brucellosis research: an evaluation. Report of the subcommittee on Brucellosis Research, National Academy of Sciences. Washington, D.C.: National Academy Press, 1977:61-77] and B. melitensis strain Rev 1 [Jimenez de Bagues, M. P. et al. (1989) Ann. Rech. Vet. 20:205-213; Pardon, P. et al. (1990) Ann. Rech. Vet. 21:153-160], are not ideal vaccine strains. Both strains cause vaccinated animals to seroconvert and thus make subsequent serological diagnosis of brucellosis difficult [Jimenez de Bagues, M. P. et al. (1992) Vet. Microbiol. 30:233-241]. Both strains can induce abortion (Jimenez de Bagues, 1989, supra; Corner, L. A. and Alton G. G. (1981) Res. Vet. Sci. 31:342-344] and both can cause disease in humans [Blasco, J. M. and R. Diaz (1993) Lancet 342:805; Young, E. J. (1983) Rev. Inf. Dis. 5:821-842]. A more recent attenuated strain of B. abortus, RB51 [Schurig, G. G. et al. (1991) Vet. Microbiol. 28:171-188], shows more promise as a live vaccine strain. RB51 is a rough strain that confers protection against infection by Brucella, yet does not cause seroconversion [Cheville, N. F. 1993, supra; Jimenez de Bagues, M. P. et al. (1994) Infect. Immun. 62:4990-4996]. However, neither the genetic basis of the RB51 rough mutation nor the basis of attenuation is known. Also, RB51 carries resistance to rifampin, an antibiotic currently used to treat brucellosis.
Therefore, there is a need for a live attenuated Brucella vaccine strain, with a defined nonreverting genetic mutation, which does not cause seroconversion in the vaccinee, and which does not retain resistance to antibiotics used in the treatment of brucellosis.
The present invention fulfills the need described above. In this application is described attenuated rough strains of Brucella, containing genetically defined mutations, which will not cause seroconversion. The mutations in these attenuated Brucella strains were created by DNA deletion, the type of mutation least susceptible to genetic reversion and are therefore advantageous as vaccine strains. These vaccines strains do not retain resistance to an antibiotic useful for treatment of brucellosis.
More specifically, this invention relates to two genetically defined rough mutants of Brucella melitensis, WRR51 and WRRP1, as candidate strains for a live vaccine against brucellosis. These strains differ from Brucella live vaccines currently used in livestock because they have genetically defined mutations that were created by deleting DNA from the Brucella chromosome. Both strains have a lipopolysaccharide (LPS) defect and thus do not cause the seroconversion that complicates disease screening. Smooth strains currently approved for use in animals are not good candidates for human vaccines because though attenuated, they can still cause disease in humans. One of the vaccine strains of the present invention, WRRP1, is a double deletion mutant that is highly attenuated and is unlikely to cause disease in humans.
Briefly, the genetically defined rough mutants of Brucella were constructed by using a Brucella abortus VTRA1 chromosome containing a Tn5 insertion which conferred a rough phenotype [Winter, A. J. et al. (1996) Amer. J. Vet. Res. 57:677-683]. The B. abortus gene containing the Tn5 insertion was cloned from the VTRA1 chromosome and the nucleotide sequence of the 2693 bp (SEQ ID NO:1) region containing the transposon insertion was determined. The Tn5 insertion was found to be located within an open reading frame of 1233 bp spanning nucleotides 883 through 2115 of SEQ ID NO:1 which coded for a gene that was distantly related (40% amino acid similarity) to the sequence of the Salmonella enterica LT2 rfbU, a gene encoding a mannosyltransferase [Liu, D. et al. (1993) J. Bacteriol. 175:3408-2414]. A deletion of 607 bp was made in the putative rfbU gene and a cassette containing a chloramphenicol acetyl transferase gene (cat) was ligated into the deletion site to create rfbU/cat. The plasmid containing rfbU/cat, pRFBU1, was electroporated into B. melitensis strain 16M and electroporants with pRFBU1 integrated were selected on Brucella agar containing chloramphenicol. Southern DNA hybridization confirmed that the chloramphenicol resistant and ampicillin sensitive electroporants had the deletion mutation carrying the chloramphenicol resistance cassette in place of the wild type chromosomal locus resulting from a directed allelic exchange by a double crossover recombinational event. The deletion strain, designated WRR51, was confirmed to be rough by staining with crystal violet, and by lack of agglutination with an anti-LPS serum.
A purE deletion was then introduced into B. melitensis strain WRR51 by a similar allelic exchange procedure. PurE is an essential enzyme in the purine biosynthetic pathway. The resultant double deletion strain (xcex94rfbU xcex94purE) was designated WRRP1. The DNA flanking the transposon insertion was sequenced to determine the open reading frame that had been interrupted to cause the rough phenotype and was found to be rfbU. The complete sequence of Brucella rfbU is described for the first time in this application in SEQ ID NO:1.
Unlike the rough mutants of the present invention, none of the rough mutants described previously including B. abortus strain 2308 rfbU mutant, VTRA1, and the VTRA1 transposon mutation integrated into the chromosomes of B. melitensis and Brucella suis by allelic exchange to create VTRM1 and VTRS1, respectively [McQuiston, J. R. et al. (1995) Abstract, CRWAD, November 1995; Winter, A. J. et al. (1996) Am. J. Vet. Res. 57:677-683] contained a defined mutation. In other words, the previously described mutant strains were produced by a transposon insertion which is a random event and can occur at any chromosomal location wherein the mutants of the present invention were produced by a directed allelic exchange to produce a unrevertable, defined deletion in the gene. A plasmid construct containing a synthetic copy of the putative rfbU gene that restored the smooth phenotype to the WRR51 deletion mutant of the present invention, did not restore the smooth phenotype to the VTRA1 transposon mutant. The inability to complement the transposon mutant indicates either that the transposon insertion confers a more general genetic defect in LPS biosynthesis (via a polar effect), or that the VTRA1 strain has additional mutations that affect LPS biosynthesis. The rough mutants of the present invention have a defined, nonreverting, deletion in the putative rfbU gene that was integrated into the chromosome by allelic exchange.
In order to construct the deletion in a rough strain, several factors had to be considered. The sequence of the flanking DNA (the rfbU gene) extending far enough in either direction of the deletion had to be known to allow for PCR or direct cloning of a large enough region of the Brucella chromosome. In addition, it was important to allow for a deletion of a significant portion of the rfbU gene to inactivate the gene in the first attempt; the actual crossover (allelic exchange) of the xcex94rfbU for the wild type was very difficult because it occurred at a very low frequency, and after several trials, it was found that a threshold of at least 500 bp on either side of the deletion was necessary for efficient homologous recombination crossover in the Brucella chromosome. High biocontainment facilities, Biosafety Level 3 (BSL3), were necessary to move the deletion construct back into Brucella to make the mutant. Introducing the deletion construct required development of a more efficient method for electroporating DNA into Brucella than used before.
Therefore, it is an object of the present invention to provide a rfbU DNA fragment encoding 2693 nucleotides useful as a diagnostic agent.
It is another object of the present invention to provide an amino acid sequence for RfbU protein encoding 411 amino acids.
It is another object of the present invention to provide a Brucella rfbU DNA fragment containing a deletion useful in attenuating a Brucella strain.
It is another object of the invention to provide a recombinant vector comprising a vector and any of the above described DNA fragments.
It is a further object of the present invention to provide a host cell transformed with any of the above-described recombinant DNA constructs.
It is another object of the present invention to provide a method for producing RfbU protein which comprises culturing a host cell under conditions such that a recombinant vector comprising a vector and the rfbU DNA fragment is expressed and RfbU protein is thereby produced, and isolating RfbU protein for use as a diagnostic agent.
It is a further object of the present invention to provide an antibody to the above-described RfbU for use as a diagnostic agent.
It is yet another object of the invention to provide a Brucella spp. vaccine comprising an attenuated rough Brucella containing a defined deletion in the rfbU gene and effective for the production of antigenic and immunogenic response resulting in the protection of an animal against brucellosis. All of the Brucella which infect humans are highly related, probably biovars of the same species [Corbel, M. J. (1997) Emerging Inf. Dis. 3:213-221]. It is expected that this live vaccine would provide cross protection against other Brucella strains since there is thought to be high homology in the rfbU gene in brucellae [Jimenez de Bagues, M. P. et al. (1994) Infect. and Immun. 62:4990-4996].
It is a further object of the invention to provide a multivalent Brucella vaccine comprising defined Brucella rfbU mutants from a variety of strains effective for the production of antigenic and immunogenic response resulting in the protection of an animal against infection with brucellae.
It is yet another object of the present invention to provide a method for the diagnosis of brucellae infection comprising the steps of:
(i) contacting a sample from an individual suspected of having the infection with antibodies which recognize RfbU protein; and
(ii) detecting the presence or absence of a complex formed between RfbU and antibodies specific therefor.
It is yet another object of the present invention to provide a method for the diagnosis of Brucella in a sample using the polymerase chain reaction, said method comprising:
(i) extracting DNA from the sample;
(ii) contacting said DNA with
(a) at least four nucleotide triphosphates,
(b) a primer that hybridizes to rfbU DNA, and
(c) an enzyme with polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of said first primer by said enzyme, whereby a first DNA product is synthesized with said DNA as a template therefor, such that a duplex molecule is formed;
(iii) denaturing said duplex to release said first DNA product from said DNA;
(iv) contacting said first DNA product with a reaction mixture comprising:
(a) at least four nucleotide triphosphates,
(b) a second primer that hybridizes to said first DNA, and
(c) an enzyme with polynucleotide synthetic activity,
under conditions suitable for the hybridization and extension of said second primer by said enzyme, whereby a second DNA product is synthesized with said first DNA as a template therefor, such that a duplex molecule is formed;
(v) denaturing said second DNA product from said first DNA product;
(vi) repeating steps iii-vi for a sufficient number of times to achieve linear production of said first and second DNA products;
(vii) fractionating said first and second DNA products generated from said rfbU DNA; and
(viii) detecting said fractionated products for the presence or absence of rfbU in a sample.
It is yet another object of the present invention to provide a method for the detection of Brucella spp. in a sample which comprises assaying for the presence or absence of rfbU RNA or DNA in a sample by hybridization assays.
It is a further object of the present invention to provide a diagnostic kit comprising a RfbU antibody and ancillary reagents suitable for use in detecting the presence of brucellae in mammalian tissue or serum.
It is a further object of the present invention to provide a diagnostic kit comprising primers specific for the amplification of rfbU sequences and ancillary reagents suitable for use in detecting the presence of brucellae in mammalian tissue or serum.
It is yet an object of the present invention to provide a therapeutic method for the treatment or amelioration of symptoms of brucellosis, said method comprising providing to an individual in need of such treatment an effective amount of sera from individuals immunized with the vaccine strains of the present invention in a pharmaceutically acceptable excipient.
It is another object of the present invention to provide a means to express antigens of interest as potential therapeutics or vaccines for human and veterinary use. RfbU is usually either cytoplasmic or associated with the inner membrane. When brucellae are lysed within host cells, RfbU and any antigen designed to be expressed with RfbU would then be accessible to the intracellular environment of the cell or host.
It is another object of the invention to provide an inactivated vaccine produced from the live attenuated Brucella described above. The attenuated Brucella of the present invention can be used in producing inactivated Brucella vaccines. By using an attenuated Brucella, particularly the double mutant which is significantly attenuated, there is a much greater margin of safety in the event that the product is incompletely inactivated. Starting with an attenuated strain is also much safer during the manufacturing phase, and may allow production under lower biocontainment levels. In addition, inactivated attenuated Brucella strains can be used to isolate subunits for subunit vaccines.